1. Field of the Invention
The present invention relates generally to an electrophoresis film support made of a microporous polymer membrane sheet, and particularly to such a film support which is particularly suited for the separation of serum proteins.
2. Prior Art Statement
The electrophoresis has been used for the separation or purification of colloidal particles or macro molecules with a net electric charge as a method particularly useful for the separation or fractionation of proteins. The known electrophoreses include, in addition to the ordinary electrophoresis, the disc-electrophoresis in which a discontinuous buffer solution system is used for effecting the aimed separation, the immuno-electrophoresis in which separation and/or detection is effected by the utilization of immuno-diffusion reactions, the isoelectric focusing method wherein a certain pH gradient is established between both electrodes so that the aimed separation or fractionation is effected in the electric field having the thus established pH gradient.
In the field of clinical test, it is needed sometimes to fractionate the serum proteins, and for this purpose, various electrophoresis film supports made of microporous films mainly composed of polymer materials have been developed and used.
For example, such a microporous polymer film support film made of cellulose acetate is used in a method for the electrophoretic fractionation of serum proteins, the method comprising the steps of dipping the microporous film support in a buffer having a concentration of 0.03 to 0.08 mole/1 and pH value of 8.6, spotting the serum on the surface of the microporous film, applying a DC current through the film to subject the proteins to electrophoresis for fractionating the same, and finally dyeing the separated proteins. Typical dyes used for dyeing the proteins include Ponceau-3R, Ponceau-S, Nigrosine, Amido Black and Coomassie-brilliant Blue. In general, the figure of the thus fractionated and dyed proteins includes five fractions which are referred to as the albumin fraction, the .alpha..sub.1 -globulin fraction, the .alpha..sub.2 -globulin fraction, the .beta.-globulin fraction and the .gamma.-globulin fraction.
The serum proteins are fractionated into five fractions, as described above. However, it is to be noted that each of these five fractions is not composed of a single chemical species but includes those which have incidentally the same mobility but have utterly different chemical structures. For example, the .beta.-globulin fraction contains co-existent LDL (low density lipoprotein) which is a complex of choresterol (free chresterol and ester-form choresterol) and apo-protein (Content: about 18% to about 22% in dry weight base), other than the globulin component.
On the other hand, the .alpha..sub.2 -globulin fraction contains VLDL (very low density lipoprotein) which is a complex of choresterol (free chresterol and ester-form choresterol) and apo-protein (Content: about 6% to about 10% in dry weight base), other than the globulin component.
The fractionation is not a method by which the serum proteins are fractionated or separated into five series of fractions simply and clearly in respect of their chemical structures, but has been carried out, as a first or primary screening step for various clinical tests, to separate the serum proteins into five fractions to ascertain that the result of fractionation pattern is not significantly different from the result of fractionation pattern of normal serum proteins. It is an ordinary practice that a further precise examination on the serum proteins should be carried out when an abnormality is found by the test result of the primary screening, i.e. observation on the five fractions.
In recent years, the results of various examinations are often stored in a computer and read by putting out the data in the digital or analog fashion. Although such an automatic processing method has a merit that the data can be read to diagnose the condition of a particular patient more precisely, it has such disadvantages that confusion in pattern recognition by the automatic preocessing might be lead due to appearance of a superfluous peak or irregularization of the pitches or spacings between adjacent peaks which might be caused even by slight change in composition of the serum proteins to be fractionated into five fractions, leading to erroneous test result. Accordingly, there is a demand for the development of an electrophoresis film support having an ability for fractionating almost all of various serums into five fractions which may be easily identified, when the film support is used for the primary screening purpose, nonetheless more or less change in composition or irregularily of peak pitches might be present.
On the other hand, the positions of VLDL and LDL (commonly referred to as "pre-.beta.-lipoprotein and .beta.-lipoprotein, respectively) in the fractionation chart are shifted depending on the contents thereof. For example, a .beta.-lipoprotein fraction (the sixth fraction) appears between the .alpha..sub.2 -globulin fraction (the third fraction) and the .beta.-globulin fraction (the fourth fraction), and the position of this .beta.-lipoprotein fraction is shifted depending on the content of .beta.-lipoprotein within the spacing between the .alpha..sub.2 -globulin fraction (the third fraction) an .beta.-globulin fraction (the fourth fraction). In detail, when the content of the .beta.-lipoprotein is small the .beta.-lipoprotein fraction is contained in the .alpha..sub.2 -glubulin fraction, but the same forms an independent peak between the .alpha..sub.2 -globulin fraction and the .beta.-globulin fraction when the content of .beta.-lipoprotein is large. Appearance of such sixth fraction hinders the analysis of the test result, since the test is normally based on the fractionation chart including five fractions.
The such hindrance on the analysis or diagnosis could be partially solved by the provision of an electrophoresis film support composed of a microporous polymer membrane sheet having a pore size distribution of from 0.1 .mu.m to 2.0 .mu.m disclosed, for example, by Unexamined Japanese Patent Publication No. 262549/1988 (Chemical Abstracts, 111: 112010v (1989)), or the provision of an electrophoresis film support composed of a microporous polymer membrane containing less than 20%, based on the weight of the polymer, of a particular wetting agent or plasticizer as disclosed by Unexamined Japanese Patent Publication No. 262550/1988 (Chemical Abstracts, 111: 112009b (1989)). However, the problem has not been fully solved by the use of the film supports disclosed by the prior publications. In addition, the problem of irregularity between the adjacent peaks of separated protein fractions has not been solved by the film supports of the publications referred to above.